The major intracellular inhibitor in human platelet activation is cAMP. The levels of this autocoid are a resultant of its rate of synthesis by adenylate cyclase and its rate of degradation by cAMP-phosphodiesterases (cAMP-PDEs). Two forms of this enzyme are potentially important in platelets: the low Km, cGMP-inhibited cAMP-PDE and the allosteric cGMP- stimulated PDE. Rationally designed inhibitors could be useful anti- platelet agents to prevent or treat arterial thrombotic events. The goal of this investigation is to delineate the structure-function correlates of platelet cAMP-PDEs. Our studies of the low Km cGMP-inhibited cAMP-PDE have identified two important functional domains. The affinity label 8-[(4- bromo-2,3-dioxobutyl)thio]adenosine-3',5'-cyclic monophosphate (8-BDB- TcAMP) irreversibly inactivated the low Km cAMP-PDE from human platelets. The substrates, cAMP and cGMP, and the product AMP protected the PDE against inactivation by 8-BDB-TcAMP indicating that the inactivation was at the active site. The incorporation of this affinity label will be quantified and correlated with the degree of inactivation. Modified peptides will be isolated and the derivatized amino acid identified and amino acid sequences determined. The structure of the active site will be compared with that of cGMP-stimulated PDE which has also been purified to homogeneity. Increased levels of intracellular cAMP have been shown to stimulate the phosphorylation of low Km cAMP-PDE which increases its specific activity, thus producing a negative feedback. The kinetic mechanism of the stimulation will be studied and the phosphorylated amino acids isolated. We will ascertain whether protein kinase C also phosphorylates platelet cAMP-PDEs. We will compare the low Km cAMP-PDE with the cGMP-stimulated PDE with regard to regulation by cAMP dependent protein kinase and protein kinase C. Cloning and sequencing of the enzyme have begun to yield the amino acid sequence in which the place the amino acids and peptides deduced from the covalent modifications of the catalytic and regulatory sites. We will complete the primary sequence of low Km cGMP-inhibited cAMP-PDE. We will then perform site directed and deletion mutagenesis to confirm the importance of the residues involved in the active site and the phosphorylation site.